| Paenibacillus polymyxa(P.polymyxa),well-known for its diverse metabolic products,emerges as a promising microbial chassis with substantial applications in chemical engineering,pharmaceuticals,and agriculture.Nevertheless,the use of P.polymyxa in synthetic biology remains limited due to the absence of appropriate tools.In this study,the range of carbon substrates that P.polymyxa can utilize was first screened and determined.Then,the metabolic pathways and regulatory network corresponding to different carbon sources were explored.After accomplishing the aforementioned goals,synthetic biology tools were ultimately created for gene editing and metabolic regulation in P.polymyxa.The specific outcomes are detailed as follows:(1)Identification of carbon substrate spectrum and exploration of metabolic regulatory pathways.The results exhibited that P.polymyxa could rapidly grow on xylose,sucrose,mannose,and mannitol.The genes related to xylose metabolism are controlled by Xyl R protein.The binding site of Xyl R,ACTTA-GTTTAAGCAATAGAC-AAAGT,covering-35 region of both Pxyl R and Pxyl AB,allows the bi-directional transcription of two genes.The genes associated with sucrose metabolism have an overlapping region with the coding sequence of its regulatory factor,Scr R.The promoter region contains four Scr R binding sites with varying binding efficiency.This structure helps scr R terminate transcription and facilitates its dynamic regulation.Mannose and mannitol metabolism-related genes are activated by transcription factors Man R and Mtl R.Their regulatory modes and binding motifs are similar to those in Bacilus subtilis.(2)Development of carbon source-inducible expression components and establishment of highly efficient inducible expression systems.Xylose,sucrose,mannose and mannitol promoters were characterized by the thermophilic lipase Tr Lip derived from Roseococcus thermophilus,and all the expression systems showed good enzymatic activity after induction by the corresponding carbon source.The Pre23,obtained by modifying the strong constitutive promoters using Xyl R binding sites,was regulated by xylose and had higher e GFP fluorescence levels than the original constitutive promoter.The sucrose-induced T7 expression system,based on the sucrose promoter,showed approximately 5 folds higher expression efficiency than the constitutive system.The promoters Pmtrl and Pltrl,which were modified with the binding sites for Man R and Mtl R derived from P.polymyxa,allows the expression being initiated by mannose and mannitol respectively.Notably,the enzyme activity of Pmtrl was 4.7 folds higher than that of the endogenous promoter Pman P.(3)Construction of carbon source-inducible CRISPR gene editing tools for P.polymyxa.A CRISPR-Cas9 gene knockout system driven by the sucrose promoter achieved an efficiency of 100% in gene knockout for P.polymyxa.This system allowed rapid recovery of selection marker and continuous gene knockout.To further enhance gene editing efficiency,a sucroseinducible CRISPR-Cpf1 gene editing system was constructed.Targeting sites with PAM sequences TTTC/TTTG achieved a 100% gene editing efficiency.This system allows simultaneous editing of multiple genes and gene knock-in.(4)Validation of gene editing systems and expression systems through enhancement of acetoin and 2,3-butanediol production in P.polymyxa.The strain DT7 S was engineered by using the developed gene editing tools,and its extracellular sucrose metabolic pathway was deleted to optimize the utilization efficiency of sucrose.In addition,the carbon metabolism flux of the acetoin synthesis pathway was enhanced using the sucrose-induced T7 expression system.The production of acetoin and 2,3-butanediol increased by 77.49% and 128.65%,respectively.In case of simultaneous enhancement of acetoin and 2,3-butanediol synthetic pathways,a 89.77%increase in acetoin and a 94.08% increase in 2,3-butanediol was achieved. |
